Abstract Gene correction therapy is one of the most important application directions in regenerative medicine. Emerging technologies such as CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated protein 9), Zinc Finger Nuclease (ZFN), and Transcription Activator-Like Effector Nuclease (TALEN) have enabled efficient and precise gene editing in a wide spectrum of species, and hold promises for eventually achieving gene correction/therapy in therapeutic settings. However, several major challenges remain to be addressed, including low knock-in efficiency, off-targeting effect and lack of an efficient delivery system in vivo. The present proposal focuses on the challenge of low knock-in efficiency. Recently we reported that RS-1, a homology directed repair (HDR) enhancer improves the efficiency of Cas9 or TALEN mediated knock-in in rabbit embryos. Microinjecting human RAD51 (hRAD51) mRNA to the embryos mimicked the beneficial effects of RS-1 treatment. In the present project, we propose experiments to further improve nuclease mediated HR rates. In Aim 1, we will first develop a RAD51 augmentation method to improve Cas9 mediated HR. On RAD51, Threonine 13 (T13) and Serine 14 (S14) are the two best known sites that are phosphorylated/activated in DNA repair processes. So we hypothesize that the replacement of T13 and S14 with their phosphomimetics (T13E and S14D) and the use of such mutant RAD51 mRNAs will lead to consecutively active RAD51 which leads to enhanced Cas9-mediated HR rate. BRCA2 is a key player in HR. It is recruited to processed double strand breaks (DSBs), and facilitates the assembly of RAD51. In Aim 2, we will develop a TALE and BRCA2 exon27 fusion protein (TALE-BE27) to help recruiting RAD51 at the DSB to further improve the HR rate. In Aim 3, we will validate these HR improving methods in rabbit embryos. The proposal aims to address a bottleneck problem in regenerative medicine (i.e. low knock-in efficiency). Its success will have significant impacts on the entire field, as a majority of stem cell based therapy will require targeted gene modifications.